Previously, complete cDNAs and complete nucleotide sequences were obtained for nine of the ten known viral mRNAs of human respiratory syncytial virus (RSV) strain A2. Here, synthetic oligodeoxynucleotides were used to direct dideoxynucleotide sequencing of intergenic and flanking regions in the viral genome (vRNA). Comparison of the intergenic and flanking sequences with the complete mRNA sequences established unambiguously the 3' to 5' order of the nine genes on vRNA, 3'-NS1-NS2-N-P-M-SH-G-F-22K-L. Each gene was immediately followed (in genome-sense) by an oligo U tract of 4-7 residues that might direct synthesis of poly A tails of the mRNAs by a reiterative copying mechanism. The intergenic regions varied in length from 1 to 52 nucleotides and displayed no obvious sequence conservation except that in all cases the last nucleotide (vRNA-sense) was an A residue. The sequence of the first 1010 nucleotides of the L gene was determined by dideoxynucleotide of sequencing of vRNA. Mapping and sequencing of the 5' end of the L mRNA established that, unexpectedly, the L gene overlaps with its upstream neighbor, the 22K gene. Specifically, the first 68 nucleotides of the L mRNA are identical to the last 68 nucleotides of the 22K mRNA, and are encoded by the same vRNA sequence. This genetic arrangement is without precedent among negative strand viruses. Further analysis of the transcription products representing this vRNA region is described. Finally, cDNA cloning and sequence analysis have been initiated for two additional RSV strains, the Long and 18537 strains. These have been shown by others to be distinct from the previously characterized A2 strain on the basis of reactivity with a panel of monoclonal antibodies to proteins of the envelope and nucleocapsid. Information on the structural and antigenic heterogeneity of RSV will be important in designing appropriate vaccine strategies.